Designed by: jeremy armetta   Group: iGEM17_Evry_Paris-Saclay   (2017-10-27)

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Applications of BBa_K2448023

Our team constructed based on this UBC 8 different highly specific biosensors: a fructose biosensor (BBa_K2448032) and 7 psicose biosensors (BBa_K2448025, BBa_K2448026, BBa_K2448027, BBa_K2448028, BBa_K2448029, BBa_K2448030 and BBa_K2448031), each of them with a particular dynamic range and sensitivity.

User Manual

To build a biosensor, you will need to insert a promoter (Figure 2) and its associated transcription factor (Figure 3) using the Golden Gate Assembly or traditional digestion-ligation.

If you use Golden Gate, the UBC uses two different type IIS restriction enzymes, BbsI for the insertion of the promoter and BsmBI for the insertion of the transcription factor. Using these enzymes for this purpose allow the manipulator to use the more common BsaI for some other experiments like, for example, the insertion of the UBC into another construct, with a simple PCR reaction.

In order to insert your transcription factor and promoters, you need to have compatible fusion sites on it, hence 5’-TGGA and GCAG-3’, and a BbsI (for promoters) or BsmBI (for transcription factors) site (Figures 2 and 3). If you use synthesized DNA, you will have to anticipate this. If you didn’t or if you use DNA you didn’t get synthesized yourself, you can easily get fusion and restriction sites with a PCR and the right set of primers.

T--Evry Paris-Saclay--cloning promoters UBC.png

Figure 2. Principle of the insertion of promoters in UBC.

T--Evry Paris-Saclay--cloning transcription factors UBC.png

Figure 3: Principle of the insertion of transcription factors in UBC.

Inserting the promoter first is recommended for two reasons:

  • The insertion marker (LacZ-alpha) allows the manipulator to see dark-green or yellow-green colonies (Figure 4) depending on whether the UBC is carrying your promoter (the yellow-green colonies) or not (the dark-green colonies). If the transcription factor is inserted first, the manipulator will have to distinguish blue colonies from dark-green ones, which can be difficult.
  • Inserting the promoter first, generally a short DNA sequence, allows you to readily see the size variation of a colony PCR product on an agarose gel because the overall construct is still short. To observe a hundred-base pair variation in a 3 or 4 kb construction can represent a greater challenge.

Note: Colonies carrying the UBC with an inserted promoter can range from yellow green to brown on IPTG+X-Gal plates, depending on the inserted promoter strength.

T--Evry Paris-Saclay--cloning promoters UBC plate.png

Figure 4. Picture of a plate containing colonies transformed with a Golden Gate reaction mixture. The Golden Gate assembly, performed on the UBC, aimed to replace LacZ-alpha by a promoter. We can clearly see on the picture a majority of yellow-green colonies (with the promoter) and some dark-green ones (with LacZ-alpha).

User Reviews

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